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,2
* Alltech France, 2–4 Avenue du 6 juin 1944, 95 190 Goussainville, France;
and
Probiotech Inc., 105 rue Echo;St-Eustache, QC, J703W3, Canada; and
and
ENESAD, 26 Bd Dr. Petitjean, BP 87 999, 21 079 Dijon Cedex, France
2 Correspondence:
phone: 33 3 80 77 25 59; fax: 33 3 80 77 25 84; E-mail:
v.julliand{at}enesad.fr.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Key Words: Cellulolytic Microorganisms • Horses • Large Intestine Fermentation • Saccharomyces cerevisiae • Starch Digestion
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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This paper presents a study designed to investigate two dietary formulations for limiting the negative impact of a starch overload (i.e., 3.4 g•kg-1BW•meal-1). The study measured the combined effects of two NDF/starch ratio in the diet and the supplementation with a live yeast culture preparation (YEA SACC1026, Alltech, Dunboyne, Ireland) on the equine lower gut microflora and their metabolic activities.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Eight resting, crossbred male mature horses (mean age 12 yr; mean BW 305 ± 18.4 kg) were allotted into pairs consisting of one cecum-and right ventral colon-fistulated animal and one cecum-fistulated animal (polyvinyl chloride cannula, i.d. 30 mm). Animals were surgically prepared by a certified large animal veterinarian, using the technique described in Drogoul et al. (2000a). The project was conducted under license from the Department of Health and Animal Care of the French Veterinary Authority. Pairs were randomly assigned to a replicated 4 x 4 Latin square experiment to evaluate two different dietary NDF/starch ratios fed daily without or with 10 g of a Saccharomyces cerevisiae (SC) preparation (YEA SACC1026, Alltech, Dunboyne, Ireland) per animal.
The feedstuffs as well as their physical form were chosen to mimic French feeding practices conducted in horse barns. Each diet was formulated using either high fiber (HF) or high starch (HS) concentrates plus wheat straw in long form (Table 1
). The ingredients of both HF and HS concentrates were ground through a 1.5 mm sieve and compressed into 3 mm diameter pellets. The HF diet contained 45.9% dehydrated alfalfa, whereas the HS diet contained 44.8% barley (Table 1). Both the HF and HS diets were fed at the same level of intake (i.e., 21.5 g•kg-1•d-1) to avoid any effect of the size of meal on the rate of passage of digesta (Drogoul et al., 2001; Pearson et al., 2001) and to provide a similar amount of crude protein (Table 1). The HS diet was formulated to allow a starch intake of 3.4 g•kg-1BW•meal-1, in order to reach the upper limit of prececal starch digestion (Potter et al., 1992). The dietary NDF to starch ratio varied by a factor of 4 (3.5 vs 1.0 respectively for HF and HS diets). Therefore, the HF diet provided 120% of French energy requirement of horses (Martin-Rosset et al., 1994), whereas the HS diet provided 160%.
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1 (5'CAAATTCACCTATATCTCA3') and 2 (5'GTGGATTTTTATTCCAACA3') primers (Ness et al., 1993). It has been deposited and registered in an official culture collection under the following reference: CBS 493.94. The SC preparation has been registered as an additive in European Union: Commission Regulation (EC) No. 1436/98 (OJ L-191, 7.7.1998, p 15) under the number 5. The enumeration of the preparation used for the trial corresponded to 4.5 x 109 cfu/g. Feeding and Management
The horses were maintained indoor in individual free-stalls bedded with flax shavings (ECOlit, Croissanville, France). They had access to a paddock 10 h/wk. Water and a block of trace-mineralized salt were offered free-choice. A double dose of Pyrantel (STRONGID, Laboratoire Pfizer, Orsay, France) followed a week later by a double dose of Ivermectin (EQVALAN, Laboratoire Merial, Lyon, France) were orally administered 15 d prior the beginning of the trial to control a wide range of gastrointestinal parasites (Herd, 1992; Hutchens et al., 1999).
The horses were weighed on two consecutive days before each diet adaptation period to adjust the feed allowance to their metabolic body weight (BW0.75). For both diets, animals were offered two equal meals (0800 and 1800) of 900 g DM of concentrate and 175 g DM of wheat straw per 100 kg BW each (Table 1
). The wheat straw was fed 30 min after each pelleted meal. The SC preparation was top-dressed on the pellets (5 g per meal).
Experimental Period and Sampling
Each period consisted of 21 d of adaptation to the diet and 14 d of sampling. For microbial analyses, intestinal contents were collected anaerobically (Julliand et al., 1999), 4 h after the morning meal on d 22 from the cecum and colon and repeated on d 26 for the colon only. Samples held at 38°C in a water bath were diluted under O2-free CO2 in an anaerobic mineral solution (Bryant and Burkey, 1953). For the first experimental period, live yeast cells were counted in both the cecum and colon from subsamples collected on d 22 from four horses fitted with cannula in the cecum and the right ventral colon. Because of the negligible amount of yeast cells in the intestinal content of horses not supplemented with the SC preparation (i.e., yeast count < 103 cfu/g), yeast counts in the subsequent three periods were determined only for subsamples from horses fed the SC supplemented diets. For biochemical measurements, a flexible tube (Cristallo extra, i.d.10 mm; length 1.2 m) was inserted into each cannula of each animal, before the morning meal on d 33 of each period, as described in Drogoul et al. (2000b). Intestinal fluid contents were withdrawn hourly during the first 12 h after feeding. Three subsamples of the filtered intestinal contents were immediately frozen (-20°C) for later analyses of concentrations of lactic acid (1 mL), VFA [(1 mL) added with a preservative (0.1 mL of a mixture of 5% H3PO4 + 1% HgCl2)] and ammonia (3 mL).
Microbial Analyses
Concentrations of Total Viable Anaerobic Bacteria. Concentrations were determined with a modified complete agar medium (Leedle and Hespell, 1980; Julliand et al., 1999), after 96 h of incubation at 38°C, on four replicate roll tubes prepared with dilutions representing 10-6, 10-7, or 10-8 mL of intestinal contents.
Concentrations of Cellulolytic Bacteria. Concentrations were determined with a modified (Baruc et al., 1983) broth medium (Halliwell and Bryant, 1963; Julliand et al., 1999), after 15 d of incubation at 38°C, as the most probable number on four roll tubes inoculated with dilutions representing 10-5, 10-6, and 10-7 mL of intestinal contents.
Concentrations of Lactic Acid-Utilizing Bacteria. Concentrations were determined with a selective medium (Mackie and Health, 1979), after 96 h of incubation at 38°C, on four replicate roll tubes prepared with dilutions representing 10-5, 10-6, or 10-7 mL of intestinal contents.
Concentrations of Streptococci spp. Concentrations were enumerated using an overlay method with a bile esculin azide agar medium (BK158HA, Biokar diagnostics, Beauvais, France). Three replicate Petri plates, prepared with dilutions representing 10-5,10-6, or 10-7 mL of intestinal contents, were counted after 48 h of incubation at 38°C.
Concentrations of Lactobacilli spp. Concentrations were determined using an overlay method with a Rogosa agar medium (BK033, Biokar diagnostics, Beauvais, France). Three replicate Petri plates, prepared from dilutions representing 10-5, 10-6, or 10-7 mL of contents, were counted after 48 h of incubation at 38°C.
Concentrations of Saccharomyces cerevisiae. This enumeration was performed in another laboratory (Sigmo, Vertou, France). Thirty millimeters of digestive samples were diluted with 270 mL sterile water and homogenized 3 min in a stomacher (Stomacher 400 LAB BLENDER, Seward Medical, London, United Kingdom). Live yeast cells were counted using a Sabouraud medium (BK025HA, Biokar diagnostic, Beauvais, France) modified with an ethanol (95%) solution plus 0.17% (vol/vol) of chloramphenicol (C0378, Sigma, Strasbourg, France). Three replicate Petri plates, prepared from each dilution representing10-3 to 10-7 mL of contents, were counted after 48 h of incubation at 35°C.
Biochemical Analyses
Cecal and colonic pH were measured immediately after each collection, using an electronic pH meter (MP120, Mettler, Toledo, Spain). Ammonia concentration was measured with a NH3 electrode (15 230 3000, Crison, Barcelona, Spain) connected to an Iono meter (GLP 22, Crison, Barcelona, Spain). Lactic acid and VFA concentrations were assayed, respectively, with an enzymatic reaction procedure (Lactate 735-10, Sigma, Strasbourg, France) quantified spectrophotometrically at 540 nm (MRX revelation, Dynatech Laboratories, Guyancourt, France) and by a gas-liquid chromatography (Gas chromatograph model 437 A, United Technologies Packard, Zurich, Switzerland) (Jouany, 1982). A ratio of acetate plus butyrate to propionate ([(A + B)/P]) was calculated according to Sauvant et al. (1994).
Statistical Analysis
The GLM procedures of SAS (SAS Inst. Inc., Cary, NC) were used to analyze the data. Logarithmic transformations were performed on microbial counts before statistical analysis. Differences in bacterial counts were tested with a model including horse as a randomized effect, diet and SC supplementation as fixed effects and the interaction between these two latter factors. A repeated measures analysis of variance was performed to compare the profiles of fermentation parameters (VFA, lactic acid, NH3 concentrations, and pH) using the repeated time option. The effect of postfeeding time was found to be significant for most of the parameters, therefore, biochemical data were analyzed for each hour individually in the bar charts. The standard deviation of the 13 sampling times after feeding was calculated for each horse and each fermentation parameter then tested with the model previously reported for bacterial data. Least squares means were calculated for all variables and separated using the pairwise t-tests (PDIFF option of SAS). All statistical procedures were performed separately for the cecum and the colon with animal as experimental unit and the significance threshold for all tests was set at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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)No live yeast cell was detected at the lowest dilution (10-3 mL) prepared from the cecal and colonic contents of horses receiving both the HF + 0 and HS + 0 treatments. Concentrations of live yeast cells increased (P < 0.01) in both the cecum and colon of horses receiving the SC preparation and averaged 4.3 x 106 and 4.5 x 104 cfu/g, respectively (data not shown).
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). However, a significant interaction between diet and SC (P < 0.01) was found for lactobacilli concentration: a positive effect (P < 0.05) of SC was registered on this specific group of bacteria when the HF diet was fed (Table 2). The HS diet increased (P < 0.001) the concentrations of total anaerobic, lactic acid-utilizing bacteria, lactobacilli, and streptococci in the cecum. On the contrary, the number of cellulolytic bacteria in the cecum decreased (P < 0.05) with the HS diet (Table 2). Although not statistically compared, the standard deviation was higher with HS + 0 than with HF + 0 in the cecum of the eight different horses, except for the streptococci. The standard deviations of total anaerobic, lactic acid-utilizing bacteria, and lactobacilli concentrations in the cecum had lower values, when SC was added in the HS diet.
In the colon, a significant (P < 0.05) interaction between diet and SC was observed for total anaerobes: the SC supplementation decreased (P < 0.05) their concentration with the HF diet. The concentrations of streptococci and lactobacilli were lower (P < 0.05) in the colon when HF diet was fed. A decrease (P < 0.05) of streptococci counts was found with the SC supplementation for the HF diet. The standard deviations of bacterial enumerations we observed, but did not analyze statistically, were different between HF and HS diets. The standard deviations of the bacterial counts in the colon appeared to decrease with the SC addition when the HF diet was fed (Table 2).
Biochemical Parameters
In the cecum, the fermentation parameters reported in Table 3 depended on the sampling time after feeding. Time interacted with the effect of the diet except for ammonia concentration and molar percentage of butyrate. Time interacted with SC supplementation for pH and lactic acid concentration.
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). The rate and extent of the pH drop was more emphasized with the HS + 0 than with the HF + 0 treatments (Figure 1). The HS diet increased (P < 0.05) the standard deviation of the cecal pH (Table 4). Saccharomyces cerevisiae supplementation increased (P < 0.05) the mean cecal pH value (Table 3) and the pH at 4, 6, and 8 h after feeding, with the HS diet (Figure 1). Additionally, the standard deviation of cecal pH decreased (P < 0.01) with the SC supplementation when the HS diet was fed (Table 4).
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). In parallel, standard deviation of the lactic acid concentration increased (P < 0.01) when the horses received the HS diet (Table 4). The mean cecal lactic acid concentration of horses fed the HS diets decreased (P < 0.05) from 407.7 to 207.5 mg/L when SC was added (Table 3). This decreasing effect appeared (P < 0.05) at 2 h until 10 h postfeeding (Figure 2). On both diets, standard deviation of the lactic acid concentration was lower (P < 0.05) in the cecum of horses supplemented with SC than that of unsupplemented horses (Table 4).
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). Saccharomyces cerevisiae supplementation decreased (P < 0.05) the mean cecal concentration of ammonia (Table 3) and the concentration during the first 5 h postfeeding (data not shown) when horses were fed the HS diet but had no clear effect with HF diet. The standard deviation of the mean concentration of ammonia decreased (P < 0.05) in the cecum with the supplementation of SC in both diets (Table 4).
Total VFA were not affected by dietary treatments. The overall concentration of acetate was reduced in the cecum (P < 0.001), while the propionate concentration increased (P < 0.001) when the horses were fed HS diets (Table 3). The postfeeding [(A + B)/P] ratio decreased (P < 0.05) from 4.37 with the HF + 0, to 3.06 with the HS + 0 treatment in the cecum (Table 3), whereas the standard deviation of this ratio increased (P < 0.05) (Table 4). When the SC preparation was supplemented in the HF and the HS diets, the molar percentage of acetate increased (P < 0.05) in the cecum of horses (Table 3).
In the colon, sampling time after feeding affected (P < 0.01) pH and the concentrations of ammonia, total VFA, acetate, and propionate. Time interacted (P < 0.01) with the effect of diet for pH and propionate concentration. There was no interaction between time and SC supplementation. The changes in the profile of the colonic pH (Figure 3) was similar to the changes found in the cecum: HS diet increased (P< 0.001) the standard deviation of the colonic pH (Table 4). No effect of SC supplementation was reported on the overall pH value, but SC decreased (P < 0.05) the standard deviation of pH when the HS diet was fed.
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). The standard deviation of the lactic acid concentration increased (P < 0.01) in the colon when the horses received the HS diet (Table 4). When SC was added, the postfeeding lactic acid concentration in the colon of horses fed the HS diet decreased (P < 0.05) from 303.2 to 235.7 mg/L (Table 3), and the standard deviation was lower (P < 0.05) (Table 4).
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). The molar percentage of acetate increased (P < 0.05) when the SC preparation was supplemented in the HS diet (Table 3). Saccharomyces cerevisiae supplementation increased (P < 0.05) the colonic [(A + B)/P] ratio (4.27 vs 4.94) in horses fed the HF diet. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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In previous works (Willard et al., 1977; Goodson et al., 1988; Julliand et al., 2001), horses were fed at the same level of energy, which induced a significant decrease in the fiber amount in starchy diets. In the present study, both the high-fiber and high-starch diets were fed at the same level of dry matter intake. Therefore, the quantity of NDF provided by the high-starch diet represented only 25% less NDF than the high-fiber diet (i.e., 660 vs 882 g of DMI/100 kg BW). In contrast, the intake of starch (252 vs 670 g of DMI/100 kg BW for HF and HS diets, respectively) increased by 166% with the high-starch diet. The larger quantity of starch provided with the HS diet allowed a greater amount of rapidly fermentable carbohydrates to reach the hindgut (Potter et al., 1992; Kienzle, 1994). Then, nondegraded starch was available for starch-utilizing bacteria (Garner et al., 1978; Goodson et al., 1988), such as lactobacilli and streptococci, and increased (P < 0.001) their concentrations in the intestinal contents. A 334% increase in the streptococci to total anaerobes ratio and a 66% decrease in the ratio of bacteria involved in the metabolism of lactic acid [i.e., lactic acid-utilizing bacteria/(lactobacilli + streptococci)] were noted in the colon of horses receiving the high-starch diet (data not shown). However, Julliand et al. (2001) reported a 95% decrease in the latter ratio, when the level of grain intake increased at the expense of fiber intake. Starch was readily used by streptococci and lactobacilli to produce lactic acid at low pH (Rowe et al., 1994; Garner et al., 1978). With the HS diet, the lowest pH (6.43) appeared 5 h after feeding, in the hindgut. At this time, the peak concentration of lactic acid (498 mg/L) in the large intestine was four times higher in the high-starch treatment than the one observed (123 mg/L) in horses fed the high-fiber diet. However, this increase in the concentration of lactic acid in equine hindgut was twice as low as that observed when the overload of starch was not balanced with a great amount of fiber (Julliand et al., 2001). The cecal concentration of cellulolytic bacteria decreased 4 h after feeding the HS diets compared to the HF diets, which was consistent with previous results reported by Garner et al., (1978) and Julliand et al., (2001). Although the mean concentration of total VFA did not differ between the two diets, we measured a significant increase in the concentration of propionate and a significant decrease in acetate concentration in the cecum and the colon when the HS diet was given to horses. Such an evolution resulted in a significant decrease of the [(acetate + butyrate)/propionate] ratio, suggesting a decline in the fibrolytic activity and an increase of the fermentation of starch in the large intestine of horses (Sauvant et al., 1994).
Viable yeast cells were found in the large intestine of supplemented horses 4 h after feeding. The concentration of yeast cells in the cecum remained at a level close to that initially supplemented (i.e., 106 cfu/g DM of feedstuff), which was in agreement with the results observed in the rumen (Durand-Chaucheyras et al., 1998; Fiems et al., 1993) and the ileum (Newbold et al., 1990) of sheep. However, only 4.5 x 104 cfu/g of viable yeast cells were detected in the colon contents. These data suggested that the strain of S. cerevisiae contained in the SC preparation was able to reach and survive in the cecum and the right ventral colon of the horse but not to colonize them. The supplementation of S. cerevisiae appeared to significantly modify pH, concentrations of lactic acid and ammonia, and molar percentages of acetate and butyrate when the high-starch diet was fed and [(acetate + butyrate)/propionate] ratio with the high-fiber diet. The interaction effect between S. cerevisiae and diet was greater inside the cecum than inside the colon. This was possibly related to the greater counts of yeast cells in the cecum than in the colon and suggests that the effects of S. cerevisiae are possible only if viable yeast cells are present in intestinal ecosytems (Nagaraja et al., 1997). With the high-starch diet supplemented with live yeast culture, the overall pH values increased, and its standard deviation decreased in the cecum. Such a buffering effect had been previously reported in ponies fed a 60:40 forage:grain ratio (Moore and Newman, 1993). Interestingly, the effect of S. cerevisiae on the cecal pH appeared from 4 to 9 h after feeding the high-starch diet, during a period when both pH values and lactic acid concentration were mainly affected by the diet. This was combined with an increase in the lactic acid-utilizing to lactic acid-producing bacteria ratio. The molar percentage of acetate was increased in the cecum and the colon with both SC-supplemented diets. This suggested an enhancement of the fibrolytic activity in the hindgut of horses supplemented with S. cerevisiae, although we did not observe any increase in the concentration of cellulolytic bacteria. This contrasts with the results reported by Moore et al. (1994), who found an increase in the cellulolytic bacterial concentration with the supplementation of S. cerevisiae. More studies are required to deeply understand the direct effects of the live yeast culture preparation on the lower gut microflora and their metabolic activities in horses.
The large standard deviations for microbial and biochemical parameters found in our study indicate the wide variation in the intestinal ecosystems of horses. This variation was greatest for horses fed the high-starch diet, which is consistent with the reports of Julliand et al. (2001) and Glinsky et al. (1976). Therefore, practical nutritional recommendations should consider differences in the ability of horses to tolerate high-starch diets to minimize the occurrence of digestive disorders. Feeding S. cerevisiae in high-starch diets reduced variation in lactic acid concentration and pH of intestinal contents suggesting that yeast supplementation may allow some horses to better tolerate high-starch diets without developing digestive disorders.
Our investigation into the effects of a large supply of starchy feeds balanced with an equivalent amount of fiber (i.e., dietary NDF/starch = 1) on the complex interactions between dietary nutrients and enteric microflora provided some useful information on the relation between nutrition and health of the equine lower gut. However, further studies are needed to evaluate this feeding practice on the cellulolytic activity of the equine digestive ecosystem and its consequences on the total apparent digestibility of the ration.
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Implications |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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Footnotes |
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Received for publication August 13, 2001. Accepted for publication May 14, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionImplicationsLiterature Cited |
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